Microscope system

ABSTRACT

A microscope system comprises: a stage carrying a sample; an optical system forming an sample image; a driver driving at least the optical system or stage to relatively moves the sample and optical system; an imaging section capturing a reference viewing field image as an image of a predetermined viewing field range of the sample and peripheral viewing field images each being an image of a peripheral viewing field range containing a predetermined region in the predetermined viewing field range and different from the predetermined viewing field range, by the driver moving the relative position of the sample; a correction gain calculator calculating a correction gain of each pixel of the reference viewing field image based on the reference viewing field image and peripheral viewing field image; and a corrector performing shading correction on the reference viewing field image based on the calculated correction gain.

TECHNICAL FIELD

The present invention relates to a microscope system.

BACKGROUND ART

A virtual slide is known in which pieces of partial image data obtainedby imaging respective parts of a sample on a slide glass at a highresolution using a microscope are connected to obtain image data of anentire sample, so that such image data can be displayed on a display ofPC or the like as a microscope image for observing.

When an image is captured with a microscope, shading occurs which meansunevenness in luminosity caused by unevenness in light source,non-uniformity of an optical system, an issue of an imaging element, orthe like. Where shading occurs, the more distant the place is from theoptical axis, it becomes darker. As a result, in a case where pieces ofpartial image data are connected together like a virtual slide, failuremay occur such as unnatural border generated at a border portion betweenpieces of partial image data, or a shading that itself looks like apattern of a sample.

To cope with this, a method is known which involves acquiring a patternof shading as a calibration image in advance and performing correctionbased on the calibration image. Patent literature 1 describes amicroscope system for performing such correction, in which image datafor calibration is obtained by escaping a sample when observing intransmission illumination and by reflecting light when observing inepi-illumination, so that, even if illumination unevenness changes whenillumination light is switched to change shading, the shading can besuppressed. Further, patent literature 2 describes a fluorescence imageacquiring device which performs imaging with a uniform fluorescencesample as a calibration sample at the time of fluorescence observation.

CITATION LIST Patent Literature

-   {PTL 1}-   Japanese Unexamined Patent Application, Publication No. 2006-171213-   {PTL 2}-   Japanese Unexamined Patent Application, Publication No. 2008-51773

SUMMARY OF INVENTION Technical Problem

However, the microscope system described in patent literature 1 may becumbersome to use because a sample under observation is required to betemporarily removed to obtain image data for calibration. Further, in acase where a calibration sample is used according to patent literature2, it is required that the calibration sample is not damaged nor duststicks to it, which makes management of the calibration samplecumbersome. Furthermore, it is difficult to generate a uniformfluorescence sample that is appropriate for a calibration sample,otherwise, desired correction may not be attained.

Solution to Problem

The present invention provides a microscope system comprising: a stageon which a sample is placed; an optical system for forming an image ofthe sample; a drive section which drives at least one of the opticalsystem and the stage to move the sample and the optical system relativeto each other; an imaging section which captures a reference viewingfield image that is an image of a predetermined viewing field range ofthe sample, and captures a plurality of peripheral viewing field imageseach of which is an image of a peripheral viewing field range thatcontains a predetermined region in the predetermined viewing field rangeand different from the predetermined viewing field range, by causing thedrive section to move a position of the sample relative to the opticalsystem; a correction gain calculation section which calculates acorrection gain of each pixel of the reference viewing field image basedon the reference viewing field image and the peripheral viewing fieldimage; and a correction section which performs shading correction on thereference viewing field image based on the correction gain calculated bythe correction gain calculation section.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an overall configuration diagram representing a microscopesystem in a first embodiment of the present invention;

FIG. 2 is an elevation view of a stage of the microscope system in oneembodiment of the present invention;

FIG. 3 is an explanatory view representing examples of a referenceviewing field image and a peripheral viewing field image;

FIG. 4 is a flow chart about shading correction in the microscope systemin the first embodiment of the present invention;

FIG. 5 is an explanatory view representing examples of the referenceviewing field image and the peripheral viewing field image;

FIG. 6 is an explanatory view representing examples of the referenceviewing field image and the peripheral viewing field image;

FIG. 7 is an explanatory view representing examples of the referenceviewing field image and the peripheral viewing field image;

FIG. 8 is an explanatory view representing examples of the referenceviewing field image and the peripheral viewing field image;

FIG. 9 is an explanatory view representing examples of the referenceviewing field image and the peripheral viewing field image;

FIG. 10 is an overall configuration diagram representing a microscopesystem in a second embodiment of the present invention;

FIG. 11 is a diagram representing a cumulative histogram in whichhistogram of brightness is integrated;

FIG. 12 is an overall configuration diagram representing a microscopesystem in a third embodiment of the present invention;

FIG. 13 is a flow chart about shading correction of a microscope systemin a third embodiment of the present invention; and

FIG. 14 is an explanatory view representing examples of the referenceviewing field image and the peripheral viewing field image.

DESCRIPTION OF EMBODIMENTS First Embodiment

A microscope system in a first embodiment of the present invention isdescribed below in reference to a figure.

As shown in FIG. 1, the microscope system includes a microscope device 1for observing a sample (specimen) A, an imaging device 2 which capturesan observation image of the sample A obtained by the microscope device1, a control section 8 for controlling the microscope device 1 and theimaging device 2, and an image processing section 9 which applies apredetermined process on an image obtained with the imaging device 2.

The illumination light emitted from a light source 7 of the microscopedevice 1 illuminates the sample A set on a stage 4 through a condenserunit 6, and an image of the sample A is focused on an imaging plane ofthe imaging device 2 by way of an objective lens 5 and a focusing lenswhich is not shown in a figure (optical system).

The stage 4 of the microscope device 1 is driven by a drive mechanismcontrol section 12 which will be stated below, thereby the sample A ismoved relative to the optical system including the objective lens 5 andthe focusing lens. FIG. 2 is an elevation view in which the stage 4 islooked down upon from the objective lens 5 side. As shown in FIG. 2, thestage 4 is secured to a holder 902, and a stepping motor is connected tothe holder 902 using a ball screw which is not shown in a figure.

The stage 4 is driven in XY direction (direction orthogonal to theoptical axis of the objective lens 5) in FIG. 2 when the drive mechanismcontrol section 12 drives the stepping motor. As a result, the sample Ais moved relative to the optical system, and furthermore relative to theimaging device 2. By moving the sample A and the imaging device 2relative to each other, a position which is illuminated by the lightfrom the light source 7 that passes through an illumination opening 901and an image that is focused on the imaging element of the imagingdevice 2 are moved.

Note that a means (actuator) for driving the stage 4 is not limited to aball screw and a stepping motor, and an ultrasonic motor, for example,may be used.

Further, the drive mechanism control section 12 controls drive positionby informing drive coordinates to an operator, which is available byimage matching such as template matching from an image obtained with theimaging device 2. It should be noted that a way of acquisition of thedrive coordinates is not limited to image matching, and a value of thescale mounted on the stage 4, for example, may be used.

The imaging device 2 is a digital camera equipped with an imagingelement such as CCD, and CMOS, and it forms a focused image and outputsthe image as a digital image to the control section 8. The imagingdevice 2 is controlled by an imaging device control section 11 which isdescribed later, for capturing a reference viewing field image (see FIG.3(A)) which is an image of a predetermined viewing field range of thesample A. It also captures a plurality of peripheral viewing fieldimages (see FIG. 3(B) to FIG. 3(E)) each of which is an image ofperipheral viewing field range that contains a predetermined region atthe center of the predetermined viewing field range and different fromthe predetermined viewing field range by causing the drive mechanismcontrol section 12 to drive the stage 4 so that the position of a sampleA is moved relative to the optical system.

The control section 8 includes the imaging device control section 11which controls the imaging device 2 and the drive mechanism controlsection 12 that controls the stage 4. The control section controls theimaging device 2 through the imaging device control section 11 and thestage 4 through the drive mechanism control section 12 respectively, soas to acquire images at a predetermined position in predeterminednumbers. More specifically, it acquires a reference viewing field imagewhich is an image of the predetermined viewing field range, and aplurality of peripheral viewing field images each of which is an imageof peripheral viewing field ranges that contains a predetermined regionat the center of the predetermined viewing field range and differentfrom the predetermined viewing field range, and then outputs the imagesthat have been obtained to an image processing section 9.

The image processing section 9 includes a correction gain calculationsection 13 and a shading correction section 14. The correction gaincalculation section 13, based on the reference viewing field image andthe peripheral viewing field image that have been inputted from thecontrol section 8, calculates a correction gain for each pixel of thereference viewing field image. The shading correction section 14,according to the correction gain available from the correction gaincalculation section 13, performs shading correction on the referenceviewing field image to acquire an image in which shading has beencorrected.

Note that, shading means unevenness in luminosity caused by unevennessin light source, non-uniformity of optical system, an imaging element ofimaging device or the like, and the shading correction means correctingof unevenness like these.

Hereinafter, flow of shading correction in the microscope system of thepresent invention is described according to a flow chart in FIG. 4. Inone embodiment, the stage 4 can move a position of the sample A relativeto an optical system, or the imaging device 2, by 1/3 each in heightdirection (Y direction) and width direction (X direction) of thereference viewing field image. Here, the imaging device 2 captures fourperipheral viewing field images that are images of peripheral viewingfield ranges that contain a predetermined range at the center of thepredetermined viewing field range and different from the predeterminedviewing field range by deviation of ⅓ each at least in one of heightdirection and width direction.

In step 201, a reference viewing field image of a predetermined viewingfield range is captured. In step 202, the stage 4 is driven to move thesample A and the imaging device 2 relative to each other so that aviewing field range becomes different. Returning to step 201 again, aperipheral viewing field image is captured. In step 202, the stage ismoved again to provide a different viewing field image for a peripheralviewing field image. Then returning to step 201 again, a peripheralviewing field image is captured. By repeating the procedure describedabove, one reference viewing field image and four peripheral viewingfield images are captured.

To be specific, in FIG. 3(A) to FIG. 3(E), 301 denotes a referenceviewing field image (predetermined viewing field range), and 302, 303,304, and 305 respectively denote peripheral viewing field images(peripheral viewing field ranges). In FIG. 3(B) to FIG. 3(E), thereference viewing field image 301 is shown with dotted line against therespective peripheral viewing field images 302, 303, 304, and 305.

In other words, after the reference viewing field image 301 is captured(FIG. 3(A)), the stage 4 is driven rightward by 1/3 of the image widthof the reference viewing field image, to image the peripheral viewingimage 302 (FIG. 3(B)). The stage 4 is driven by 1/3 of the image heightof the reference viewing field image downward and also by 1/3 of theimage width leftward, to image the peripheral viewing field image 303(FIG. 3(C)). The stage 4 is driven downward by ⅓ of the image height ofthe reference viewing field image, to image the peripheral viewing fieldimage 304 (FIG. 3(D)). Lastly, the stage 4 is driven rightward by 1/3 ofthe image width of the reference viewing field image and by 1/3 of theimage height downward, to image the peripheral viewing field image 305(FIG. 3(E)). Thus imaging is completed in step 203 and a flow proceedsto next step 204.

Note that an order of imaging respective peripheral viewing field imagesis optional, and the reference viewing field image 301 and theperipheral viewing field images 302-305 may be in opposite relativepositional relationship.

In step 204 to step 207, alignment with a reference viewing field image,brightness image conversion, low-pass filter process, and correctiongain calculation are performed for each of all the peripheral viewingfield images.

Note that, as shown in FIG. 5, the height and width of each image isdivided in three sections for convenience sake, and each image includesa central region and eight peripheral regions which surround the centralregion, with each of height and width being 1/3. As an example, a region401 shown in FIG. 5(A) is a central region of the reference viewingfield image 301 and corresponds to a peripheral region of the peripheralviewing field image 303, and further, a region 402 is a peripheralregion of the reference viewing field image 301 and corresponds to acentral region of the peripheral viewing field image 303.

In step 204, the reference viewing field image is aligned with any oneof the peripheral viewing field images. In short, by the driving methodand the imaging method of the stage 4 described above, the referenceviewing field image 301 and the peripheral viewing field image 303 arecaptured theoretically at the position shown in FIG. 5(A). However,deviation may occur because of issue of alignment accuracy or resolutionfor the stage 4. To cope with this, a scale value of the stage 4, thepulse number of a stepping motor, execution of matching using images, orcombination of them is used for adjusting position, to allow a pixel ofthe reference viewing field image and that of the peripheral viewingfield image to be associated with each other, for alignment.

Then, in next step 205, brightness image conversion is performed in acase where the reference viewing field image and the peripheral viewingfield image are color images. In the aforementioned brightnessconversion, RGB value may be subjected to weight addition, or anarbitrary value in RGB may be selected. In step 206, an image afterbrightness conversion is applied with a low-pass filter process. Bythis, an effect of alignment error or deviation caused by aberration maybe corrected.

In short, such a problem is solved as, in a case where accuratepositioning cannot be attained by alignment or in a case where alignmentcannot be performed because of effect of distortion aberration or comaaberration, a structure will appear in a gain if the gain is calculatedas it is. Since shading is generally present in a low frequency range, alow-pass filter is applied to suppress occurrence of structurecomponent. Application of a low-pass filter may include application ofconvolution, for example, convolution of Gaussian filter in imageprocessing or application of an average filter. It may also includeapplication of a bandpass filter in a frequency range in which an imageis Fourier transformed.

In next step 207, a correction gain is calculated with reference to FIG.5(B) and FIG. 6. FIG. 6(F), for convenience sake, exemplifies an image507 to have the same size and shape as the reference viewing field imageor the peripheral viewing field image divided in three-by-threesections. A correction gain is calculated for peripheral regions A-Iexcept for a central region E of the image 507.

The flow of correction gain calculation is to be explained with aperipheral viewing field image 303 as an example. As described above, aregion 401 is a central region of the reference viewing field image 301and corresponds to a peripheral region of the peripheral viewing fieldimage 303, and a region 402 is a peripheral region of the referenceviewing field image 301 and corresponds to a central region of theperipheral viewing field image 303.

Accordingly, the region 401 of the peripheral viewing field image 303 isused to calculate a correction gain of a region C of the division image507, and the region 402 of the peripheral viewing field image 303 isused to calculate a correction gain of a region G in the division image507.

It is assumed that height of a reference viewing field image is H, widthis W, and the upper left of an image is origin, while the right side isX coordinate and the lower side is Y coordinate respectively. Here, acorrection gain of the region C is to be calculated. With brightnessvalue of coordinates (x′, y′) of the reference viewing field image 301assumed as f (x′, y′) and brightness value of coordinates (x, y)corresponding to (x′, y′) of the peripheral viewing field image 303 as g(x, y), coordinates of the reference viewing field image 301 becomes(x′, y′)=(x−W/3, y+H/3). A gain GainC (x, y) of the region C is obtainedthrough a formula (1) below.Gain_(C)(x,y)=f(x′,y′)/g(x,y)  (1)

Further, a correction gain of the region G is to be calculated. Withbrightness value of coordinates (x′, y′) of the reference viewing fieldimage 301 assumed as f (x′, y′) and brightness value of coordinates (x,y) corresponding to (x′, y′) of the peripheral viewing field image 303as g (x, y), coordinates of the reference viewing field image 301becomes (x′, y′)=(x+W/3, y−H/3). A gain GainG (x, y) of the region C isobtained through a formula (2) below.Gain_(G)(x,y)=f(x′,y′)/g(x,y)  (2)

Accordingly, correction gain of each pixel in regions 401 and 402 of thereference viewing field image, in other words, the division region C andthe division region G are calculated based on a ratio between thebrightness of the peripheral region 401 of one peripheral viewing fieldimage 303 which overlaps with the reference viewing field image 301 andthe brightness of the central region 401 of the reference viewing fieldimage, and a ratio between the brightness of the peripheral region 402of the reference viewing field image 301 that overlaps with oneperipheral viewing field image 303 and the brightness of the centralregion 402 of the peripheral viewing field image 303.

In a similar procedure, a correction gain of two regions can becalculated for one peripheral viewing field region. Further, byperforming the alignment in step 204 to the calculation of correctiongain in step 207 on each peripheral viewing field region, a correctiongain can be calculated for A-I regions except for the region E. It is tobe noted that the region E is a central region and, therefore, allcorrection gains are 1, and as a result, correction gains for the pixelsare determined in all regions of the reference viewing field image. Step208 determines that all of regions have been completed, and a flowproceeds to next step 209.

In step 209, seam correction is performed. The seam correction is tomake a border portion smooth. It is performed because picture qualitydegrades, if mismatching occurs to be noticeable at a border among theregions A-I that are used for convenience sake for calculation of thecorrection gain described above. The seam correction is attained byapplying a low-pass filter such as Gaussian filter for smooth connectionat the seam.

Note that in step 207 described above, a region that has been set forcalculating correction gain is set to be 1/3 of height and width of thereference viewing field image, however weight synchronization may beapplied with change as shown in FIG. 7. To be specific, in FIG. 7, aregion is not divided in three but they are set larger, and as a result,regions are calculated to overlap each other as shown with a shadowarea. An overlapped region 601 is used for correction.

Further, FIG. 8(A) shows a correction gain in a case in which a centralregion is set to be larger than a drive pitch. In that case, correctiongains appear in overlapped manner near a border 602. By applying aweight depending on position to a correction gain near the border 602for synthesization according to a graph shown in FIG. 8(B), mismatchingnear the border can be corrected. Further, a seam may be smoothed byusing a low-pass filter instead of setting a central region to be largerthan a drive pitch.

A correction gain for the entire image is obtained through the processesstated above. As a result, in step 210, an image in which shading hasbeen corrected is obtained by multiplying a reference viewing fieldimage with a correction gain. Furthermore, it is possible to hold acorrection gain, and it may be then applied to an image that has beenobtained, a live image, or an image for image connection.

As described above, in a microscope system of the embodiment, areference viewing field image and a plurality of peripheral viewingfield images each of which contains a predetermined central region ofthe reference viewing field image and which have a different viewingfield range from each other are captured. Based on the images, acorrection gain is calculated for each pixel of the reference viewingfield image, and based on the correction gain, the reference viewingfield image is applied with shading correction. As a result, it is notrequired that a sample is escaped out of a field angle or calibrationsample dedicated for shading correction is prepared. Consequentlyshading correction is made with ease and sure.

In short, the imaging device captures the reference viewing field imagewhich is an image of a sample in a predetermined viewing field range,and further captures a plurality of peripheral viewing field images aswell by driving any one or both of the optical system and the stage byusing an imaging device control section or a drive mechanism controlsection, which mean drive sections, so that a position of the sample ismoved relative to the optical system. Note that, the center of the imageobtained by the imaging device is a region that is brightest in theimage because it almost matches with an optical axis, therefor thatregion causes no shading issue. However, an outside region of the imageis a dark region which is away from the optical axis, and therefore theregion has a shading issue.

To cope with this, as peripheral viewing field images, a plurality ofimages of peripheral viewing field ranges are captured which contain apredetermined region in a predetermined viewing field range anddifferent from other predetermined viewing field ranges. Then, based onthe reference viewing field image and the peripheral viewing fieldimages, a correction gain of each pixel of the reference viewing fieldimage is calculated, and after that based on the correction gain, thereference viewing field image is applied with shading correction. Thisway, shading correction is performed with ease and sure.

Note that, it is preferred that the imaging device captures a pluralityof peripheral viewing field images each of which is an images ofperipheral viewing field range containing a predetermined region at thecenter of the predetermined viewing field range and different from thepredetermined viewing field range. As described above, the center ofimage is the brightest region because the center almost matches with theoptical axis. As a result, that region has no shading issue. For thisreason, shading correction is efficiently performed by allowing aplurality of peripheral viewing field images to include a predeterminedregion at the center of the predetermined viewing field range.

Note that, it is possible that a region, where shading does not occur,occurs away from an optical axis because of adjustment error in anoptical system or the like. In that case, prior to capturing thereference viewing field image or the peripheral viewing field image, aregion where shading does not occur is to be specified. Thespecification of the region may be performed by a user by inputting avalue in an image processing section 9 with the use of an interfacedevice such as a mouse and a keyboard, or may be done by automaticsetting according to a condition of the optical system stored in advancein the image processing section 9.

In case a region in which shading does not occur is not specified, asshown in FIG. 9, a reference viewing field image is divided by the sizeof a specified region 161 for convenience sake, and a peripheral viewingfield image is acquired in such a manner as the region 161 overlaps withall other regions in a divided image 160. Here a region division numberis assumed to be N. N pieces of peripheral viewing field images atmaximum are obtained if all regions are imaged in a case where relativepositional relationships are identical, and (N+1)/2 pieces, in minimum,of peripheral viewing field images are obtained if any one of regions isimaged in the case of identical relative positional relationships, inaddition to a reference viewing field image. Note that the selectedregion 161 is not limited to be square, and such graphic as polygon orcircle may be employed.

Note that, although FIG. 1 shows a configuration related to observationunder transmitted illumination, there is no limitation and observationunder epi-illumination may be employed.

Further, driving of the stage 4 is not limited to be performed by thedrive mechanism control section 12, and the driving may be performedmanually. Furthermore, a driving pitch of the stage 4 is not limited to1/3 the width and height of the reference viewing field image describedabove, and it may be 1/N (N is an odd number of 3 or larger). With thedrive pitch assumed to be 1/N of width and height, imaging may be onlyrepeated while driving in such a manner as a central region available bydividing an image in N×N overlaps with other regions, with a region 401and a region 402 in FIG. 5(A) being examples. As with the case of 1/3pitch described above, only (N×N+1)/2 images are required to be obtainedif second imaging is not performed when relative positions of thereference viewing field image and the peripheral viewing field image areidentical.

In short, the imaging device control section or the drive mechanismcontrol section, being a drive section, is possible to move a sampleposition relative to the optical system in height direction and widthdirection of the reference viewing field image by 1/N (N is odd number)each time, and the imaging device is preferred to image a plurality ofperipheral viewing field images each of which is an image of peripheralviewing field range that contains a predetermined range at the center ofthe predetermined viewing field range and different from other viewingfield ranges in at least one of height direction and width direction bydeviation of 1/N respectively.

With this configuration, an overlapping region of the reference viewingfield range and an interested peripheral viewing field range and aborder between them become clear in each peripheral viewing field image,thereby calculation becomes easy when calculating a correction gain.Further, even if a correction gain changes in a discontinuous mannerbetween overlapping regions because of alignment error between thereference viewing field image and the interested peripheral viewingfield image, or because of a difference in brightness between centralpredetermined regions, correction is made easily on the overlappingregion and the border. It is to be noted that the correction gain of allpixels of the reference viewing field image can be calculated bycapturing (N²−1)/2+1 peripheral viewing field images if calculation of acorrection gain is intended for regions corresponding to mutual centralpredetermined regions based on the reference viewing field image and oneperipheral viewing field image.

Not that, determination of the number of obtained images to be obtainedis not limited to use the method described above, and N×N pieces inmaximum may be captured even if relative positional relationships areidentical. In that case, although the time required for acquisitionincreases, if a drive pitch is made shorter, the size of a region usedfor correction can be reduced as well, resulting in enhanced correctioneffect. Thus, variation among samples is reduced because increase in thenumber of obtained images allows correction on the same region.

Furthermore, although the embodiment described above explains the casein which five peripheral viewing field images are obtained, nine imagesin total may be obtained which includes a reference viewing field imageand eight peripheral viewing field images, being dislocated 1/3 each,for example. Furthermore N×N images may be obtained in the case of thedrive pitch being N (N is odd number).

Second Embodiment

Hereinafter, a second embodiment of the present invention will bedescribed in reference to FIG. 10.

A microscope device applied to a microscope system related to theembodiment includes (as shown in FIG. 10), an excitation light source701 and a fluorescence cube 702. The light from the excitation lightsource 701 is radiated to a sample A on which fluorescence reagent isapplied by way of the fluorescence cube 702 and an objective lens 5. Thefluorescence emitted from the sample A is focused on an imaging plane ofthe imaging device by way of the objective lens 5, the fluorescence cube702, and a focusing lens which is not shown in a figure.

According to the microscope system described above, shading correctionis performed as will be described below. In short, in reference to theflow chart in FIG. 4 described above, alignment is performed between thereference viewing field image and the peripheral viewing field image andthen brightness image conversion is performed, and after thatdecoloration correction is made prior to processing using a low passfilter.

In the decoloration correction, dropping of fluorescence emission causedby decoloration of fluorescence reagent is corrected. A ratio betweenbrightness average values of a central region overlapping 602 as shownin FIG. 8 is used as a decoloration gain, and the gain is applied to allpixels in an interested image for correction. As a result, the change inluminosity across the entire image caused by decoloration is corrected,thereby constructing an image which has no decoloration. It should benoted that, a method of correction for decoloration is not limited tothe method described above. A method that utilizes a cumulativehistogram, for example, which is disclosed in Japanese Unexamined PatentApplication, Publication No. 2011-95073 may be applied.

A principle of decoloration correction that utilizes the cumulativehistogram is to be described in reference to FIG. 11. The FIG. 11 showsa cumulative histogram in which a brightness histogram is integrated forthe central region overlapping 602 so as not to be affected by shadingas described above. A graph 102 shows an image obtained in advance, andthe graph 101 shows an image obtained thereafter. It is understood thatdecoloration has occurred in the graph 101 and notable difference inluminosity appears near the central brightness, however, the brightnessis darkening, with little change, in low brightness region and highbrightness region.

The fact that luminosity changes depending on brightness like thisindicates that a large change caused by decoloration occurs at middlebrightness but little change occurs at low brightness because lowbrightness is a background region, and almost nothing illuminates bysuch light quantity at high brightness. A decoloration correction isattained by making correction so that the graph 101 agrees with thegraph 102. Therefore, based on the cumulative histogram 102 of referenceimage and the cumulative histogram 101 of an image to be corrected, alookup table is generated which converts the cumulative histogram 101 ofthe image to be corrected into a cumulative histogram of a referenceimage.

In other words, such lookup table is generated as the cumulativehistogram 102 of the image to be corrected, having been subjected togradation correction by using the lookup table, agrees with thecumulative histogram of the reference image. The lookup table isgenerated for each of RGB. To be specific, the lookup table satisfiesrelational expression (4) for gradation Iout of a reference image andgradation IIN of an image to be corrected in relational expression (3)shown below, where Iout is the gradation (brightness) to be a reference,Sout (Iout) is cumulative pixel numbers of reference image, IIN isgradation (brightness) of image to be corrected, SIN (IIN) is an imageto be corrected, and LUT means input/output relation which the lookuptable has.Sout(Iout)=SIN(IIN)  (3)Iout=LUT[IIN]  (4)

The gradation of an image to be corrected can be corrected using LUTgenerated as above, thereby correcting decoloration. Since a portionwith little change such as low brightness portion and high brightnessportion as described above is not enhanced, it is possible to correctonly a region where no fluorescence substance is present or a region ofdecoloration excluding self fluorescence.

As described above, the microscope system in the embodiment captures thereference viewing field image and a plurality of peripheral viewingfield images each of which contains a predetermined region at the centerof reference viewing field image and have a viewing field rangedifferent from each other. Based on the images, a correction gain iscalculated for each pixel of the reference viewing field image, andbased on the correction gain, the reference viewing field image isapplied with shading correction. As a result, a sample is not requiredto be escaped from an angle of view, nor, a special calibration sampleis required to be prepared for shading correction, resulting inperforming of shading correction with ease and sure.

In short, a correction gain calculation section calculates a correctiongain for each pixel of the reference viewing field image based on theratio between brightness of a region of the peripheral viewing fieldimage which overlaps with the reference viewing field image andbrightness of a predetermined region at the center of the predeterminedviewing field range and the ratio between brightness of a region of thereference viewing field image which overlaps with the peripheral viewingfield image and brightness of a predetermined region at the center ofthe peripheral viewing field range.

Consequently, based on the reference viewing field image and oneperipheral viewing field image, a correction gain can be calculated forregions corresponding to mutual central predetermined regions, allowingshading correction with ease and with less amount of calculation.

Third Embodiment

A third embodiment of the present invention is described below inreference to FIG. 12.

In the embodiment, a control section 8 includes a partial imageacquisition section 121, and in accordance with this, an imageprocessing section 9 includes a composite section 122. The partial imageacquisition section 121 is a means for acquiring an image forconstructing a wide viewing field image by compositing images together,and it controls a drive mechanism control section 12 for driving foracquiring a partial image so that the images acquired by controlling animaging device control section 11 overlap each other at image edges.

Such a microscope system performs shading correction as described belowaccording to a flow chart in FIG. 13. To be specific, a procedurestarting from imaging of a reference viewing field image and peripheralviewing field images down to seam correction is performed like in thefirst embodiment (see step 201 to step 209 in FIG. 4). Therefore, samesigns are attached in step 201 to step 209 and related explanation is tobe omitted. In the embodiment, after the seam correction, a partialimage is obtained by the partial image acquisition section 121.

As shown in FIG. 14, in order to obtain a partial image 141 in step 131,a partial image 2 is captured by driving a stage 4 so that it partiallyoverlaps with a partial image 1, which procedure is repeated until apartial image 9 is captured. Note that, the order of imaging and thenumber of images are not limited to this, and any order and any numberof images may be allowed.

In next step 132, a partial image that has been imaged is corrected byusing a correction gain that has been calculated. By this, shading inthe partial image is corrected, and unnaturalness is eliminated at aconnection part of partial images.

Performing of partial image acquisition and application of shadingcorrection to all partial images means completion of image acquisition133, to proceed to next step 134. In step 134, partial image compositingis performed. In short, image matching such as template matching is usedfor aligning overlap of partial images, and then synthesizing isperformed. Here, the alignment is not limited to image matching, and adriving amount of the stage 4 may be used. Further, in synthesizing,weight synthesizing as shown in FIG. 8(B) or the like may be performed.

As described above, an image compositing device in the embodiment canconstruct a composite image having been applied with shading correction,without escaping a sample nor using a dedicated calibration sample.Further, a user can obtain a composite image that has been applied withshading correction automatically with no labor instead of obtaining acalibration image before obtaining the composite image.

REFERENCE SIGNS LIST

-   1 Microscope device-   2 Imaging device-   4 Stage-   5 Objective lens-   6 Condenser unit-   7 Light source-   8 Control section-   9 Image processing section-   11 Imaging device control section-   12 Drive mechanism control section-   13 Correction gain calculation section-   14 Shading correction section

The invention claimed is:
 1. A microscope system comprising: a stageconfigured to support a sample; an optical system configured to form animage of the sample; a drive actuator that is configured to drive atleast one of the optical system and the stage and is configured to movethe sample and the optical system relative to each other; an imagingsensor configured to capture a reference viewing field image, which isan image of a viewing field range of the sample on the stage, and theimaging sensor is configured to capture a peripheral viewing fieldimage, wherein a first central region, which contains a center of theperipheral viewing field image, overlaps a first peripheral region ofthe reference viewing field image, wherein a range of peripheral viewingfield image is different from the viewing field range, wherein theimaging sensor is configured to cause the drive actuator to move aposition of the sample relative to the optical system from the viewingfield range of the sample to the range of peripheral viewing field; acorrection gain calculation section that is configured to calculate acorrection gain of each pixel in the first peripheral region of thereference viewing field image based on (1) an image of the firstperipheral region of the reference viewing field image and (2) an imageof the first central region of peripheral viewing field image; and acorrection section that is configured to perform shading correction onthe reference viewing field image based on the correction gaincalculated by the correction gain calculation section, wherein thecorrection gain calculation section is configured to calculate thecorrection gain for each pixel of the first peripheral region of thereference viewing field image based on a ratio between brightness of thefirst central region of peripheral viewing field image that overlaps thefirst peripheral region of the reference viewing field image andbrightness of the first peripheral region of the reference viewing fieldimage.
 2. The microscope system according to claim 1 wherein, the driveactuator is configured to move the position of the sample by 1/N (N isodd number) in a height direction and move the position of the sample by1/N (N is odd number) in a width direction of the reference viewingfield image relative to the optical system, and wherein the imagingsensor is configured to capture each of the plurality of peripheralviewing field images that are of different ranges from each other,wherein each of the plurality of peripheral viewing field imagesdeviates from the viewing field range at least in height direction by1/N or width direction by 1/N.
 3. The microscope system according toclaim 2, wherein the correction gain calculation section calculates acorrection gain for each of the plurality of peripheral regions of thereference viewing field image.
 4. The microscope system according toclaim 1, wherein the correction gain calculation section is configuredto calculate a first correction gain of each pixel of the firstperipheral region of the reference viewing field image based on an imageof the first peripheral region of the reference viewing field image andan image of the first central region of the peripheral viewing fieldimage, the correction gain calculation section is configured tocalculate a second correction gain of each pixel in a second peripheralregion of the reference viewing field image based on an image of asecond peripheral region of the peripheral viewing field image and animage of a second central region of the reference viewing field image,the second central region of the reference viewing field imagecontaining a center of the reference viewing field image and overlapsthe second peripheral region of the peripheral viewing field image, andthe correction section is configured to perform shading correction onthe first peripheral region and the second peripheral region of thereference viewing field image based on the first correction gain and thesecond correction gain determined by the correction gain calculationsection.